The objectives of this study were to determine genetic variation and to analyze population structure of 6 Italian local chicken breeds involved in a conservation program. Twenty microsatellite markers were investigated in 337 birds belonging to 6 breeds: Ermellinata di Rovigo, Robusta Maculata, Robusta Lionata, Pépoi, Padovana, and Polverara; a commercial layer cross was used as reference. One hundred twelve alleles were detected in the overall population, with a mean number of 5.6 +/- 2.1 alleles per locus. For the local breeds, the observed and expected heterozygosity ranged from a minimum of 0.240 to a maximum of 0.413 and from 0.243 to 0.463 for the Pépoi and Polverara breeds, respectively. Deviation from Hardy-Weinberg equilibrium was observed in 5 breeds and in the commercial cross. The overall population heterozygote deficiency was 0.427, the average inbreeding coefficient was 0.097, and the heterozygote deficiency due to breed subdivisions was 0.437. Reynolds' distances were used to draw an unrooted neighbor-joining tree, which topology gave information on the genetic origin of these breeds and confirmed their known history. The estimated molecular kinship within a breed ranged from 0.559 to 0.769, evidencing high coancestry. Structure analysis was performed to detect the presence of population substructures. Inferred clusters corresponded to the different breeds, without presence of admixture. The exception was the Polverara breed, for which a more complex genetic structure was found. The results supported the decision of safeguarding these breeds as an important reservoir of genetic diversity and confirmed the usefulness of microsatellite markers to characterize and to monitor genetic variability in local chicken breeds.